Yersinia pestis, the etiologic agent of bubonic, pneumonic, and septicemic plague, is unique among bacteria in that wild-type strains require high concentrations of Ca2+ for growth at human host temperatures (37 degrees C) but not at 26 degrees C in vitro. The physiological basis for this requirement is not known. Mutants of Y. pestis that do not require the cation are avirulent due to an inability to synthesize a specific protein, V, and a lipoprotein, W. These bacteria, since they are facultative intracellular parasites, must be able to survive both within and without the host cell. The temperature-dependent requirement for Ca2+ exhibited in vitro may reflect important physiological changes that the pathogen undergoes in order to survive in the host cell. The long term objective of my research program is to define the physiological roles that Ca2+, the V protein, and the W lipoprotein play in Y. pestis and to determine the relationship of these properties to the virulence of these pathogens. The focus of this specific proposed study is to investigate the thermoregulation of cytoplasmic membrane synthesis and function in Y. pestis and to determine the influence of Ca2+ on these processes. There are two specific areas that I intend to investigate regarding this: 1) Temperature induced changes in membrane composition, particularly in the phospholipid and fatty acid profiles of the cytoplasmic membrane, and 2) Temperature induced alterations in membrane function and permeability, particularly in the generation of a proton motive force and in the transport of amino acids. The first area will be investigated employing thin layer and gas chromatographic methodologies to define the phospholipid and fatty acid profiles under the experimental conditions. I will employ membrane filtration and flow-dialysis methodologies to determine membrane permeability and transport of selected probes under experimental conditions.